Counting of Rif1p and Rif2p on Saccharomyces cerevisiae Telomeres Regulates Telomere Length

Abstract

Telomere length is negatively regulated by proteins of the telomeric DNA-protein complex. Rap1p in Saccharomyces cerevisiae binds the telomeric TG 1-3 repeat DNA, and the Rap1p C terminus interacts with Rif1p and Rif2p. We investigated how these three proteins negatively regulate telomere length. We show that direct tethering of each Rif protein to a telomere shortens that telomere proportionally to the number of tethered molecules, similar to previously reported counting of Rap1p. Surprisingly, Rif proteins could also regulate telomere length even when the Rap1p C terminus was absent, and tethered Rap1p counting was completely dependent on the Rif proteins. Thus, Rap1p counting is in fact Rif protein counting. In genetic settings that cause telomeres to be abnormally long, tethering even a single Rif2p molecule was sufficient for maximal effectiveness in preventing the telomere overelongation. We show that a heterologous protein oligomerization domain, the mammalian PDZ domain, when fused to Rap1p can confer telomere length control. We propose that a nucleation and spreading mechanism is involved in forming the higher-order telomere structure that regulates telomere length.Essential for faithful chromosome maintenance and replication, the ends of eukaryotic chromosomes, telomeres, are dynamic entities whose structures are regulated (26, 43). In the budding yeast Saccharomyces cerevisiae, each telomeric terminal DNA region consists of a tract of irregular TG 1-3 repeat sequences onto which sequence-specific DNA binding proteins and associated proteins assemble. Telomeres in dividing yeast cells are maintained between 250 to 350 bp through a dynamic balance of lengthening and shortening activities. Telomeric DNA shortens as a result of the inability of the general DNA replication machinery to fully replicate the ends of linear DNA molecules (the "end replication problem") and nuclease action (31, 51). Telomere lengthening is primarily mediated by telomerase, a cellular reverse transcriptase that catalyzes the de novo addition of telomeric DNA to chromosome ends, using a sequence within its intrinsic RNA subunit as a template (3).How telomere structure is modulated by the telomeric DNA tract length and how length-dependent structural changes regulate the lengthening and shortening activities at the telomere are central but unresolved questions. Available data support the proposal that long telomeres assume an as-yet-unknown structural state that is inhibitory for telomerase-mediated telomere elongation, while short telomeres can undergo a structural change that results in either recruitment or activation of telomerase (4, 46). Physical models for higher-order telomere structure have been proposed. A "fold-back" model places the end of the telomere inward toward the subtelomeric region, preventing action by telomerase (11,33,45). In a related model, suggested by the clustering of telomeres at the nuclear periphery (17), telomere-telomere interactions restrict accessibility to telomerase.

Rif1 and Exo1 regulate the genomic instability following telomere losses

“…5A). Previously, it was found that Rif2 and Rif1 have synergistic effects in inhibiting telomerase (Wotton & Shore, 1997; Levy & Blackburn, 2004) and yet opposing effects in regulating the proliferation of telomere‐dysfunctional cdc13‐1 cells (Addinall et al ., 2008; Xue et al ., 2011). In conclusion, Rif1 is unique among many telomere‐associated proteins in facilitating proliferation of cells lacking telomeres, whereas Rif2 and Mre11 have the opposite effect to Rif1.…”

Palmitoylation controls the dynamics of budding-yeast heterochromatin via the telomere-binding protein Rif1

“…PFA4 was important for Rif1-GFP's localization to discrete foci at the nuclear periphery. Several studies provide evidence that these foci correspond to telomeric clusters (22,25,35). Rif1 binds telomeres via direct interactions with the telomere-binding protein Rap1, and this interaction is relevant to Rif1's molecular roles at telomeres (25,33).…”

Telomeres and telomerase: their mechanisms of action and the effects of altering their functions

“…A multi-component ''telomere homeostasis'' system prevents, on the one hand, the over-extension of telomeres. Protein-protein interactions among the telomere-associated proteins are important for this function, which acts in cis on a telomere [2].…”